Hypothalamic astrocyte NAD+ salvage pathway mediates the coupling of dietary fat overconsumption in a mouse model of obesity

Nicotinamide adenine dinucleotide (NAD)+ serves as a crucial coenzyme in numerous essential biological reactions, and its cellular availability relies on the activity of the nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed salvage pathway. Here we show that treatment with saturated fatty acids activates the NAD+ salvage pathway in hypothalamic astrocytes. Furthermore, inhibition of this pathway mitigates hypothalamic inflammation and attenuates the development of obesity in male mice fed a high-fat diet (HFD). Mechanistically, CD38 functions downstream of the NAD+ salvage pathway in hypothalamic astrocytes burdened with excess fat. The activation of the astrocytic NAMPT–NAD+–CD38 axis in response to fat overload induces proinflammatory responses in the hypothalamus. It also leads to aberrantly activated basal Ca2+ signals and compromised Ca2+ responses to metabolic hormones such as insulin, leptin, and glucagon-like peptide 1, ultimately resulting in dysfunctional hypothalamic astrocytes. Our findings highlight the significant contribution of the hypothalamic astrocytic NAD+ salvage pathway, along with its downstream CD38, to HFD-induced obesity.

Source data are provided as a Source Data file.Two-sided unpaired t-test (b, c).n.s.; not significant.Two independent replicates were performed for all studies.
Results are presented as mean ± SEM.Source data are provided as a Source Data file.Results are presented as mean ± SEM.Source data are provided as a Source Data file.

Supplementary Figure 9. Test of region specificity of astrocytes
To validate the accurate isolation of region-specific astrocytes, isolated astrocytes were subjected to qPCR analysis of brain region-specific astrocyte markers such as Lhx2, Agt, and Emx2 (n = 3 wells).One-way ANOVA followed by Fisher's LSD test.
Two independent replicates were performed.Results are presented as mean ± SEM.Source data are provided as a Source Data file.
Comparison of body weights (a) and calorie intakes (b) between hGFAP-Cre Comparison of body weights and calorie intakes between Nampt-AKO and Nampt-WT female mice following tamoxifen injection during sequential chow diet (CD) and high-fat diet (HFD) feeding conditions (n = 7 mice).c Body mass analysis in 18-week-old Nampt-AKO and Nampt-WT female mice (n = 7 mice).d, e Glucose and insulin tolerance tests (GTT, ITT) in 18-week-old Nampt-AKO and Nampt-WT female mice (n = 7 mice).f-k Energy expenditure, RER, and locomotor activity in 16-week-old Nampt-AKO and Nampt-WT female mice fed a CD (f-h) or HFD (i-k) (n = 4 mice).Two-way repeated measures ANOVA followed by Fisher's LSD test (a, d-k) and two-sided unpaired t-test (b, c).n.s.; not significant.Two independent replicates were performed for all studies.Results are presented as mean ± SEM.Source data are provided as a Source Data file.

Supplementary Figure 5 .
Effects of NAMPT modulation on NAD + contents and NAMPT enzyme activity in hypothalamic astrocytesa Body weights and fat mass of 16-week-old Nampt-AKO and Nampt-WT male mice that were subjected to hypothalamic leptin response study (n = 5 mice).b The changes in NAMPT activity and cellular NAD + contents in hypothalamic astrocytes subjected to Nampt overexpression (O/E) alone or with FK866 cotreatment (500 nM) (n = 3 wells).c Changes in cellular NAD + contents in primary cultured hypothalamic neurons and microglia treated with astrocyte conditioned medium (ACM) collected from astrocytes with or without Nampt O/E (n = 3 wells).Two-sided unpaired t-test (a) and one-way ANOVA followed by Fisher's LSD test (b, c).n.s.; not significant.Two (a, c) or three (b) independent replicates were performed.Results are presented as mean ± SEM.Source data are provided as a Source Data file.O/E) + IgG Ab ACM (Nampt O/E) + NAMPT Ab P=0.008 P=0.001 P=0.001 P=0.001 P<0.001 P<0.001 P=0.020 P=0.004 Supplementary Figure 6.Identification of the downstream mediator for the NAD + salvage pathway-activated inflammation in hypothalamic astrocytes Effects of SIRTs inhibition (a-d) and PARP inhibition (e) on Nampt overexpression (O/E)-induced inflammation in primary hypothalamic astrocytes (n = 3 wells).One-way ANOVA followed by Fisher's LSD test.Three independent replicates were performed for all studies.Results are presented as mean ± SEM.Source data are provided as a Source Data file.Metabolic phenotype analysis in mice with i.c.v.injection of 78c a Reduced hypothalamic CD38 enzyme activity in 16-week-old C57 male mice infused with 78c (n = 3 mice).b, c Calorie intakes (Vehicle, n = 8 mice; 78c, n = 7 mice) and locomotor activity (n = 5 mice) in 14~16 week-old C57 male mice during 78c treatment and HFD feding.d, e Adipocyte cell sizes analysis from H & E staining and sympathetic innervation in the BAT, iWAT, and gWAT of 16-week-old C57 male mice infused with 78c or vehicle and fed a HFD for 4 weeks (n = 5 mice).Scale bars: 500 μm.arb.unit: arbitrary unit.Two-sided unpaired t-test (a, b, d, e) and two-way repeated measures ANOVA followed by Fisher's LSD test (c).n.s.; not significant.Two (a, d, e) or three (b, c) independent replicates were performed.Results are presented as mean ± SEM.Source data are provided as a Source Data file.
of successful Cd38 knockdown in the hypothalamic astrocytes Confirmation of successful viral infection targeting Cd38 knockdown in hypothalamic astrocytes by demonstrating deletion of mCherry expression and reduced CD38 expression in hypothalamic ARH astrocytes in 16-week-old hGFAP-Cre ERT2 male mice (n = 5 mice).Arrows indicate soma of ARH astrocytes with successful viral infection, while arrowheads indicate soma of non-infected ARH astrocytes.Scale bars: 25 μm.arb.unit: arbitrary unit.Two-sided unpaired t-test.Two independent replicates were performed.